Dyslipidemia-induced endothelial dysfunction is known to play a major role in the initiation of atherosclerosis. Our studies discovered that plasma hypercholesterolemia results in suppression of endothelial inwardly-rectifying K+ (Kir) channels, one of the major endothelial ion channels and a flow sensor. Our long term goal is to elucidate the mechanisms responsible for cholesterol-induced regulation of endothelial ion channels and determine the contribution of these mechanisms to endothelial dysfunction. During the two previous funding periods of this grant, we have provided the first mechanistic insights into cholesterol-induced suppression of Kir channels and demonstrated that it correlates with an impairment of flow-induced vasodilatation in vivo. In the current proposal, we extend these studies to address three new goals: In Aim 1, we elucidate further the molecular basis of cholesterol-induced suppression of Kir channels specifically focusing on identifying putative cholesterol binding sites and determining how cholesterol binding regulates channel gating. To achieve this goal, we will use a combination of Molecular Dynamics simulations, a state-of-the-art computational approach, with site-directed mutagenesis, and biophysical and biochemical approaches to determine the impact of these mutations on cholesterol sensitivity of Kir channels and on cholesterol-Kir binding. The analysis will be done for an array of different sterols that differ in their ability to bind to the channels and/or affect channel function. In Aim 2, we will extend our studies to determine the sensitivity of endothelial Kir channels to pro- and anti-atherogenic lipoprotein profiles and hemodynamic environments and test the impact of cholesterol-induced suppression of endothelial Kir channels on the imbalance between NO and ROS production. Specifically, we will test the hypothesis that Kir channels are suppressed by pro-atherogenic lipoproteins (LDL, oxLDL) and flow environment (disturbed flow) and rescued by anti-atherogenic lipoproteins (HDL) and laminar unidirectional flow. Furthermore, we will address the hypothesis that suppression of endothelial Kir results in the inhibition of NO release and increase in ROS production and that this mechanism contributes significantly to endothelial dysfunction. Finally, in Aim 3, we will test the hypothesis that cholesterol-induced suppression of Kir channels plays a major role in the impairment of flow-induced vasodilatation in isolated arteries, a hallmark of endothelial dysfunction. Specifically, we will first test the role of Kir channels in endothelial dysfunction in ApoE-/- knockout mice, one of the well-established animal models of atherosclerosis and then we will extend our studies to human arteries isolated from biopsies obtained from patients with pro (high LDL) and anti (high HDL)- atherogenic lipoprotein profiles. Molecular techniques will be employed in human arterial tissue to determine the mechanism of Kir channels contribution to flow induced vasodilatation a critical endothelium- dependent mechanism of blood flow regulation. We believe that taken together, these studies will make a significant contribution to the understanding of cholesterol regulation of ion channels and dyslipidemia- induced endothelial dysfunction.